Human anaplasmosis caused by Anaplasma phagocytophilum (Ap) is now the second most prevalent tick-borne illness in North America, with increasing incidence also in Europe and Asia. The acute, pro-inflammatory syndrome is characterized by high fever, pancytopenia and elevated serum transaminases, requiring hospitalization in >36% of patients, with an overall mortality rate of ~1%. A major impediment to understanding the mechanisms whereby these obligate intracellular bacteria cause disease has been the lack of reproducible systems for mutagenesis to study gene function. This has been partially overcome with the establishment of a library containing ~1,200 intra- and intergenic insertions using human cells to select mutants. The broad, long-term goals of this renewal application are to use the principles of functional genomics to understand how Ap bacteria use their genome and specific genes to thrive in two biologically vastly different hosts, mammals and ticks, and cause human disease. Our central hypothesis is that many mutations that are tolerated in an in vitro human cell culture system will nevertheless produce a deficient phenotype in human and tick cells in vitro, and in mice and ticks. Therefore, we 1) plan to catalogue and develop the currently uncharacterized library into a resource for the scientific community. We will test the hypothesis that mutants selected in a human promyelocyte cell line will allow detailed analyses of the molecular basis of gene function in cell cultures from humans and ticks. We propose a high throughput screen of candidate mutants for defective infectivity in mice and ticks, and analyses for changes in phenotype in vitro using human and tick cell culture systems. 2) We will use advanced imaging technology, proteomics and 2-hybrid bacterial screens to reveal intracellular trafficking of secreted effectors and their interactions with host targets, with emphasis on T4SS structures and effectors, other secreted proteins, outer membrane proteins and hypothetical proteins that were selected using bioinformatics-based predictions. 3) We hypothesize that mutants selected in vector tick cell culture will additionally identify genes that are essential for growth in human cells, and which are not represented in the current library. To do this, we will refine methods for selection of transposon mutants in tick cel culture using constructs designed for facile complementation of mutants with wild-type gene sequences to restore function, and develop site-directed mutagenesis using CRISPR/Cas9 mediated allelic exchange. Investigators from three collaborating laboratories will join in this effort to maximize advancement of research plans. The knowledge gained will broadly impact the field of rickettsiology and identify the molecular basis underlying pathogenic mechanisms and survival strategies of arthropod-borne intracellular bacteria. These efforts will enable a gian step forward in functional genomics and enable rational design of vaccines and drugs for the Rickettsiales, a group that includes severe pathogens, e.g., Ap, Ehrlichia chaffeensis, Rickettsia rickettsii and R. prowazekii for which such tools are either lacking or in great need of refinement